Kinetic evidence of the existence of a regulatory phosphoenolpyruvate binding site in maize leaf phosphoenolpyruvate carboxylase.
Phenylphosphate, a structural analog of phosphoenolpyruvate (PEP), was found to be an activator of phosphoenolpyruvate carboxylase (PEP carboxylase) purified from maize leaves. This finding suggested the presence in the enzyme of a regulatory site, to which PEP could bind. We carried out kinetic studies on this enzyme using controlled concentrations of free PEP and of Mg-PEP complex and developed a theoretical kinetic model of the reaction. In summary, the main conclusions drawn from our results, and taken as assumptions of the model, were the following: (i) The affinity of the active site for the complex Mg-PEP is much higher than that for free PEP and Mg2+ ions, and therefore it can be considered that the preferential substrate of the PEP-catalyzed reaction is Mg-PEP. (ii) The enzyme has a regulatory site specific for free PEP, to which Mg2+ ions can not bind. (iii) The binding of free PEP, or an analog molecule, to this regulatory site yields a modified enzyme that has much lower apparent Km values and apparent Vmax values than the unmodified enzyme. So, free PEP behaves as an excellent activator of the reaction at subsaturating substrate concentrations, and as an inhibitor at saturating substrate concentrations. These findings may have important physiological implications on the regulation of the PEP carboxylase in vivo activity and, consequently, of the C4 pathway, since increased reaction rates would be obtained when the concentration of PEP rises, even at limiting Mg2+ concentrations.
Rodríguez-Sotres
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Departamento de Bioquímica, Facultad de Química, UNAM, Mexico.
Muñoz-Clares
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D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology
D010728:Phosphoenolpyruvate; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase